Rita Sahar

Sarin-induced neuropathology in rats

Sarin, a highly toxic cholinesterase (ChE) inhibitor, administered at near 1 LD50 dose causes severe signs of toxic cholinergic hyperactivity in both the peripheral and central nervous systems (CNS). The present study evaluated acute and long-term neuropathology following exposure to a single LD50 dose of sarin and compared it to lesions caused by equipotent doses of soman described previously. Rats surviving 1 LD50 dose of sarin (95 micrograms/kg; IM), were sacrificed at different time intervals post exposure (4 h-90 days) and their brains were taken for histological and morphometric study. Lesions of varying degrees of severity were found in about 70% of the animals, mainly in the hippocampus, piriform cortex, and thalamus. The damage was exacerbated with time and at three months post exposure, it extended to regions which were not initially affected. Morphometric analysis revealed a significant decline in the area of CA1 and CA3 hippocampal cells as well as in the number of CA1 cells. The neuropathological findings, although generally similar to those described following 1 LD50 soman, differed in some features, unique to each compound, for example, frontal cortex damage was specific to soman poisoning. It is concluded that sarin has a potent acute and long-term central neurotoxicity, which must be considered in the design of therapeutic regimes.

Ocular injuries following sulfur mustard exposure: Pathological mechanism and potential therapy

Sulfur mustard (SM) is a potent vesicant, known for its ability to cause incapacitation and prolonged injuries to the eyes, skin and respiratory system. The toxic ocular events following sulfur mustard exposure are characterized by several stages: photophobia starting a few hours after exposure, an acute injury phase characterized by inflammation of the anterior segment and corneal erosions and a delayed phase appearing following a clinically silent period (years in human). The late injury appeared in part of the exposed eyes, expressed by epithelial defects and corneal neovascularization (NV), that lead to vision deficits and even blindness. During the last years we have characterized the temporal development of ocular lesions following SM vapor exposure in rabbits and have shown the existence of two sub-populations of corneas, those exhibiting delayed ocular lesions (clinically impaired) and those exhibiting only minor injuries if at all (clinically non-impaired). The aim of the present study was to investigate the pathological mechanism underlying the delayed injury by focusing on the unique characteristics of each sub-population and to test the efficacy of potential treatments. Clinically impaired corneas were characterized by chronic inflammation, increased matrix metalloproteinase (MMP) activity, poor innervation and limbal damage. Moreover, using impression cytology and histology, we identified the delayed lesions as typical for an ocular surface disorder under the category of limbal epithelial stem cell deficiency (LSCD). These results point to therapeutic directions, using anti-inflammatory drugs, MMPs inhibitors, neurotrophic factors and amniotic membrane transplantation. Topical anti-inflammatory drugs, either steroid (Dexamycin, DEX) or non-steroidal anti-infllammatory drug (NSAID, Voltaren Ophtha) were found to be beneficial in ameliorating the initial inflammatory response and in postponing the development of corneal NV, when given during the first week after exposure. When DEX was administered as a symptomatic treatment against NV, a significant regression in the angiogenic process was observed, however, the effect was temporal and blood vessels reappeared after therapy ceased. Chronic administration (8 weeks) of the MMP inhibitor Doxycycline was also effective in attenuation of the acute and delayed injury. Preliminary results, using amniotic membrane transplantation revealed some decrease of corneal edema with no effect on corneal NV. It is suggested that the chronic inflammation and prolonged impairment of corneal innervation are playing a role in the pathogenesis of the delayed LSCD following SM exposure by creating a pathological microenvironment to limbal epithelial stem cells, thus, leading to their slow death and to a second cascade of pathological events eventually resulting in severe long-term injuries. As of today, only topical anti-inflammatory drugs reached the criteria of an applicable efficient post-exposure ocular treatment for SM injuries. Further studies are required to investigate the effects of SM on epithelial stem cells and their involvement in the pathogenesis of the long-term injuries.

Characterization of acute and delayed ocular lesions induced by sulfur mustard in rabbits

Purpose. To establish an experimental model for sulfur mustard-induced acute and delayed ocular lesions in rabbits. Methods. Rabbit eyes were exposed to sulfur mustard (HD) vapor (370, 420 µg/l) for a period of two minutes. A three months follow-up study was carried out, based on the evaluation of clinical, biochemical and histological parameters. Results. HD exposure initiated typical clinical symptoms within 2–6 hrs, characterized by eye closure, eyelid swelling, conjunctival hyperemia, corneal erosions and inflammation. The clinical signs were significantly dose-dependent and reached a peak at 24–72 hrs post exposure. Biochemical evaluation of the aqueous humor exhibited an inflammatory reaction and oxidative stress at 4 hrs after exposure, subsiding at 28 hrs after exposure. Histological examination of corneas at 48 hrs revealed epithelial denudation and marked stromal edema, accompanied by cellular infiltration. Epithelial regeneration started after 72 hrs, and recovery was almost completed within 1Ð2 weeks, depending on the HD dose. A second phase of pathological processes started as early as two weeks post exposure and was characterized by corneal edema, opacity, recurrent erosions and neovascularization. The delayed injuries were found in 25 and 40% of the eyes respectively, and when appearing, were more severe than the initial ones. Conclusions. The development of HD-induced ocular lesions in rabbits is similar to the lesions described in human casualties. Quantitative analysis of the various clinical parameters emphasizes the contribution of each tissue to the overall toxic picture. Our experimental model is useful for studying the pathological mechanisms of HD-ocular lesions, and may serve for testing potential therapies.

Long-Term Study of Brain Lesions Following Soman, in Comparison to DFP and Metrazol Poisoning

The long-term histopathological effects of acute lethal (95 μg kg-1) and sublethal (56 μg kg-1) doses of soman were studied in rats and were compared to lesions caused by equipotent doses of either another cholinesterase (ChE) inhibitor, DFP (1.8 mg kg-1), or a non-organophosphorus convulsant, metrazol (100 mg kg-1). Severe toxic signs were noted following one LD50 dose administration of all the compounds, yet only soman induced brain lesions. Moreover, even when administered at a sublethal dose (0.5 LD50), soman induced some histological changes without any clinical signs of intoxication. Soman-induced brain lesions were assessed quantitatively using a computerized image analyser. The analysis was carried out for up to 3 months following administration, and a dynamic pattern of pathology was shown. The cortical thickness and area of CA1 and CA3 cells declined significantly as early as 1 week post-exposure. No pathological findings were detected following DFP and metrazol administration. It is therefore suggested that brain lesions are not common for all ChE inhibitors and that convulsions per se are not the only factor leading to brain damage following the administration of soman. The degenerative process (found also with the sublethal dose of soman) might be due to a secondary effect, unrelated to somans clinical toxicity, but leading to long-term brain injuries.

BENEFICIAL EFFECTS OF TOPICAL ANTI-INFLAMMATORY DRUGS AGAINST SULFUR MUSTARD-INDUCED OCULAR LESIONS IN RABBITS

Ocular injuries following sulfur mustard (HD) exposure are characterized by an inflammatory response, observed as eyelid swelling, conjunctivitis, corneal oedema and cellular infiltration starting 1–4 h after exposure, depending on dose. These effects heal partially during the first 1–2 weeks after exposure, with the later appearance of neovascularization, recurrent erosions and recurrent oedema of the cornea (delayed response). We have shown previously that topically applied steroid treatment, administered after HD exposure, attenuated the extent of neovascularization, one of the characteristics of delayed ocular pathology in rabbits.

Potential anti-inflammatory treatments against cutaneous sulfur mustard injury using the mouse ear vesicant model.

In spite of several decades of research, no effective treatment to skin injuries following exposure to sulfur mustard (HD) has yet been found. In the present study, the mouse ear vesicant model was applied to awake mice in order to evaluate the efficiency of potential anti-inflammatory treatments in preventing HD-induced skin damages. Clinical follow-up and histological evaluation were used to characterize the injuries to the skin and to evaluate the efficiency of the drugs that were applied. Thus, the extent of mouse ear oedema and the histopathological changes following a single application of 0.2 or 1 L of neat HD for 10 min (representing moderate and severe lesions, respectively), were monitored. Typical HD skin lesions were observed including epithelial and dermal damage. The development of the injury in mouse ears was found to be very similar to that reported in human skin. Screening of post-exposure topical steroids and non-steroidal anti inflammatory drugs (NSAIDs) proved that HD-induced inflammation could be diminished significantly as long as the treatment was applied during the early stages following exposure. A combined application of these drugs approved to be particularly effective in reducing inflammation.

Sulfur mustard toxicity in macrophages: effect of dexamethasone

Cells from the murine macrophage‐like cell line J774A.1 (J774) and cultures of primary alveolar macrophages (PAM) obtained from guinea pigs were exposed to sulfur mustard (HD, 50‐200 μM) and treated with dexamethasone (2.5 μM) 10 min after HD exposure. Cell cultures were studied at 3 and 24 h after exposure by the cleavage of Thiazolyl blue reaction (MTT) reaction and crystal violet staining (viability assays), by morphological observation and by [3H]thymidine incorporation. Exposure of J774 cells to HD caused a dose‐dependent decrease in viability that was evident at 24 h. Although no significant change in viability was observed at 3–4 h after HD exposure, a dose‐dependent decrease in [3H]thymidine incorporation was observed. Treatment with dexamethasone caused a dose‐dependent decrease in viability. However, the combined exposure to HD and dexamethasone had a synergistic effect on the decrease of cell viability. This synergistic effect is not due to a change in DNA synthesis rate because [3H]thymidine incorporation was not affected by dexamethasone. In PAM cultures, HD caused some ‘activating’ effect on [3H]thymidine incorporation and an increase in cell number at the lower dose (100 μM) but this was less at 200 μM. Both effects were reduced by dexamethasone treatment. We conclude that macrophages derived from different sources exhibit a different responsiveness to immunomodulators (HD and dexamethasone) and that dexamethasone can reduce the ‘inflammatory’ effect of HD in PAM. Published in 2000 by John Wiley & Sons, Ltd.

Protection by extracellular glutathione against sulfur mustard induced toxicity in vitro

1 The present study characterizes the role of extracellularly added glutathione in protection against sulfur mustard (HD) toxicity in a macrophage monocyte cell line J774. 2 Toxic effects of HD depend on dose and duration of exposure with an ED50 of 50 and 75 mM for dividing and confluent cells respectively. 3 Exposure to HD, 100-200 mM caused *15% decrease in the cellular glutathione (GSH) content 2 h after exposure, pretreatment with GSH, 0.2-10 mM, elevated cellular GSH *61.5. 4 GSH pretreatment increased cell viability after HD 2-3-fold. Similar protective effects of GSH treatment were found in a human epidermoid carcinoma cell line (KB). 5 Protection by post treatment with GSH was apparent even 60 min post HD exposure. 6 No protection was afforded when the intracellular GSH concentration was elevated prior to exposure and the extracellular GSH had been washed out. However, GSH depleted cells were more sensitive to HD than normal cells, and were also protected by addition of GSH to the growth medium, although the intracellular GSH content remained low. 7 We conclude that it is essential for the GSH to be present extracellularly in order to protect cells from HD toxicity. 8 Our findings have therapeutic implications in particular for the protection of lungs after inhalation exposure to HD vapor.

Characterization of acute and long-term sulfur mustard-induced skin injuries in hairless guinea-pigs using non-invasive methods

Background/purpose: Skin exposure to sulfur mustard (HD) results in erythema, edema and severe injury, which take long time to heal and might impose a heavy burden on the health system. Despite many years of research, there is no treatment that prevents the development of the cytotoxic effects of HD causing acute and prolonged damage to the skin. Therefore, it is of great importance to develop treatments that will ameliorate the extent of injury and improve as well as shorten the healing process. The aim of the present study was to establish a small animal model for a long‐term HD‐induced skin injury using the hairless guinea‐pig (HGP) and to further test the efficacy of anti‐inflammatories in ameliorating the pathology.

Delayed Loss of Corneal Epithelial Stem Cells in a Chemical Injury Model Associated with Limbal Stem Cell Deficiency in Rabbits

Purpose: Ocular injuries following exposure to the chemical agent sulfur mustard (SM) are characterized by acute corneal erosions and inflammation of the anterior segment that may be followed by delayed Partial Limbal Stem Cell Deficiency (LSCD), expressed clinically by corneal neovascularization and epithelial defects. LSCD may derive from direct destruction of limbal stem cells or indirectly from altered limbal stromal niche. The aim of this study was to investigate the mechanism underlying LSCD in SM injuries, focusing on the effects of the chemical on limbal epithelium. Methods: Rabbit eyes were exposed to SM vapor and were observed by slit lamp examinations and pachymetry. Eyes were taken for histological and molecular biology evaluations at different time points (4 h–4 weeks), to include acute and delayed injuries. Epithelial stem cells were identified by ABCG2, p63 and by in vivo BrdU labeling for slow cycling cells. Results: Limbal stem cells were not damaged during the acute phase following SM exposure, in contrast to the severe injury of the central corneal epithelium. On the contrary, limbal epithelium became activated, responding to corneal insult with a wound healing process, as shown by histology and by transient elevation of the stem cells markers. Simultaneously, inflammation was taking place in the limbal stroma lasting for weeks. A gradual loss of stem cells was observed later-on (2–4 weeks), associated with typical symptoms of LSCD. Conclusions: LSCD associated with SM ocular toxicity was not derived from a direct cytotoxic effect on the epithelial stem cells, but apparently from pathological events at the limbal stroma, that produced an abnormal microenvironment for the stem cells, triggering their gradual death. The results, and in particular the absence of a primary damage to the epithelial stem cells, indicate the presence of a therapeutic window for intervention to avoid the development of the delayed LSCD.